There is a considerable body of literature describing the potential for evolving proteins for a variety of characteristics, especially enzymes for example, to be stabilized for operation at different conditions. For example, enzymes have been evolved to be stabilized at higher temperatures, with varying activity. In situations where there is an activity improvement at the high temperature, a substantial portion of the improvement can be attributed to the higher kinetic activity commonly described by the Q10 rule where it is estimated that in the case of an enzyme the turnover doubles for every increase of 10 degrees Celsius. In addition, there exist examples of natural mutations that destabilize proteins at their normal operating conditions, such as wild-type temperature activity of the molecule. For temperature mutants, these mutants can be active at the lower temperature, but typically are active at a reduced level compared to the wild type molecules (also typically described by a reduction in activity guided by the Q10 or similar rules).
It is desirable to generate useful molecules that are conditionally activated, for example virtually inactive at wild-type conditions but are active at other than wild-type conditions at a level that is equal or better than at wild-type conditions, or that are activated or inactivated in certain microenvironments, or that are activated or inactivated over time. Besides temperature, other conditions for which the proteins can be evolved or optimized include at least pH, osmotic pressure, osmolality, oxidation and electrolyte concentration. Other desirable properties that can be optimized during evolution include chemical resistance, and proteolytic resistance.
Many strategies for evolving or engineering molecules have been published. US 2010/0189651 discloses an engineered antibody containing an antibody or antibody fragment linked with a masking moiety. Such an engineered antibody can be further coupled to a cleavable moiety, resulting in an antibody that can be conditionally activated. The cleavable moiety is capable of being cleaved, reduced, or photolysed. The antibody can exhibit a conformation such that the antibody is more accessible to a target after removal of the masking moiety by cleavage, reduction, or photolysis of the cleavable moiety.
US 2013/0101555 discloses engineered activatable proprotein compositions. An activatable proprotein contains a functional protein coupled to a peptide mask, and further coupled to an activatable linker. In a non-activated state, the peptide mask inhibits binding of the functional protein to its target or binding partner. In an activated state, the peptide mask does not inhibit binding of the functional protein to its target or binding partner. Proproteins can provide for reduced toxicity and adverse side effects that could otherwise result from binding of a functional protein at non-treatment sites if it were not inhibited from binding to its binding partner at such non-treatment sites. Proproteins containing the peptide mask can also have a longer in vivo or serum half-life than the corresponding functional protein not containing the peptide mask.
US 2011/0229489 discloses antibodies with pH dependent binding to antigens such that the affinity for antigen binding at physiological pH (i.e., pH 7.4) is greater than at endosomal pH (i.e., pH 6.0 or 5.5). Such pH-dependent antibodies preferentially dissociate from the antigen in the endosome. This can increase antibody half-life, as compared to antibodies with equivalent KDS at pH 7.4 but with no pH dependent binding, when the antigen is one that undergoes antigen-mediated clearance (e.g., PCSK9). Antibodies with pH-dependent binding can decrease total antigen half-life when the antigen undergoes reduced clearance after being bound to an antibody.
US 2013/0266579 discloses a conditionally active anti-EGFR antibody. The anti-EGFR antibody exhibits a ratio of binding activity to human epidermal growth factor receptor (EGFR) for conditions in a tumor environment to conditions in a non-tumor environment of at least 3.0. The conditions in a tumor environment comprise one or both of a pH of from 5.6 to 6.8 or a lactate concentration of from 5 mM to 20 mM, and a protein concentration from 10 mg/mL to 50 mg/mL. The conditions in a non-tumor environment comprise one or both of a pH of from 7.0 to 7.8 or a lactate concentration of from 0.5 mM to 5 mM, and a protein concentration of from 10 mg/mL to 50 mg/mL. The anti-EGFR antibody is said to be conditionally active under conditions that may be found in a tumor microenvironment.
Pardoll et al, “The blockade of immune checkpoints in cancer immunotherapy,” Nature Review Cancer, vol. 12, pages 252-264, 2012 describes a cancer therapy that involves activating host anti-tumour immunity by blockading host immune system checkpoints. Such a blockade may be achieved by inhibiting immune checkpoint proteins such as receptors on T-cells, including cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and death protein 1 (PD1). Antibodies against these immune checkpoint proteins have been developed for cancer therapy.
Engineering or evolving a protein to be inactive or virtually inactive (less than 10% activity and especially 1% activity) at its wild type operating condition, while maintaining activity equivalent or better than its wild type condition at new conditions, requires that the destabilizing mutation(s) co-exist with activity increasing mutations that do not counter the destabilizing effect. It is expected that destabilization would reduce the protein's activity greater than the effects predicted by standard rules such as Q10, therefore the ability to evolve proteins that work efficiently at lower temperature, for example, while being inactivated under their normal operating condition, creates an unexpected new class of conditionally active proteins.
Throughout this application, various publications are referenced by author and date. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the disclosure described and claimed herein.